Methods for treating multiple sclerosis employing desmethylselegiline

ABSTRACT

Methods and pharmaceutical compositions for using desmethylselegiline. In particular, the present invention provides novel compositions and methods for using desmethylselegiline for selegiline-responsive diseases and conditions. Diseases and conditions responsive to selegiline include those produced by neuronal degeneration or neuronal trauma and those due to immune system dysfunction. Desmethylselegiline is the R-(−) enantiomer of N-methyl-N-(prop-2-ynyl)-2-aminophenylpropane. Claimed compositions include both the R-(−) isomer and mixtures of the R-(−) and S(+) isomers. Pharmaceutically acceptable acid addition salts may also be used. Effective dosages are a daily dose of at least about 0.015 mg/kg of body weight.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.08/679,330, filed on Jul. 12, 1996 U.S. Pat. No. 6,348,208 U.S.application Ser. No. 08/679,330 is a continuation-in-part ofPCT/US96/01561, with an international filing date of Jan. 11, 1996, acontinuation-in-part of U.S. Provisional Application No. 60/001,979,filed Jul. 31, 1995, and a continuation-in-part of U.S. 08/372,139,filed Jan. 13, 1995, now abandoned.

FIELD OF THE INVENTION

The present invention pertains to methods and pharmaceuticalcompositions for using the selegiline metabolite R(−)desmethylselegiline(also referred to simply as “desmethylselegiline” or “R(−)DMS”) eitheralone or in combination with its enantiomer, entdesmethylselegiline(also referred to as “S(+)desmethylselegiline” or “S(+)DMS”). Inparticular, the present invention provides compositions and methods forusing these agents in the treatment of selegiline-responsive diseasesand conditions, particularly diseases or conditions involving neuronaldegeneration or neuronal rescue).

BACKGROUND OF THE INVENTION

Two distinct monoamine oxidase enzymes are known in the art: monoamineoxidase A (MAO-A) and monoamine oxidase B (MAO-B). The cDNAs encodingthese enzymes show different promoter regions and distinct exonportions, indicating they are encoded independently at different genepositions. In addition, analysis of the two proteins has showndifferences in their respective amino acid sequences.

The first compound found to selectively inhibit MAO-B wasR-(−)-N-methyl-N-(prop-2-ynyl)-2-aminophenylpropane, also known asL-(−)-deprenyl, R-(−)-deprenyl, or selegiline. Selegiline has thefollowing structural formula:

Selegiline is the active ingredient of a human drug product and is knownin the art as a component of a therapeutic package. In particular, seePhysicians Desk Reference (1995) pp. 2430-2432 (1995 PDR), describingEldepryl® Tablets, manufactured by Somerset Pharmaceutical, Inc. andmarketed by Sandoz, active ingredient of which is selegiline. Forexample, the 1995 PDR describes a 5 mg. selegiline hydrochloride tabletand further describes the manner in which selegiline-containingtherapeutic packages are supplied for commercial use or sale. Inparticular, the 1995 PDR discloses that 5.0 mg Eldepryl Tablets are soldin “NDC 39506-011-25 bottles of 60 tablets.”

In commercial use, selegiline-containing therapeutic packages arelabeled and otherwise indicated for use in Parkinsonian patientsreceiving levodopa/carbidopa therapy. The 1995 PDR cited above providesan example of the complete approved labeling that is employed in knowntherapeutic packages. Accordingly, known in the prior art aretherapeutic packages providing one or more unit doses of selegiline asan active ingredient thereof, supplied in a finished pharmaceuticalcontainer that contains said unit doses, and further contains orcomprises labeling directing the use of said package in the treatment ofa human disease or condition as described above.

The selectivity of selegiline in the inhibition of MAO-B is important toits safety profile following oral administration. Inhibition of MAO-Amay cause toxic side effects by interfering with the metabolism oftyramine. Tyramine is normally metabolized in the gastrointestinal tractby MAO-A but when MAO-A is inhibited, tyramine absorption is increasedfollowing consumption of tyramine-containing foods such as cheese, beer,herring, etc. This results in the release of catecholamines which canprecipitate a hypertensive crisis, producing the “cheese effect.” Thiseffect is characterized by Goodman and Gilman as the most serious toxiceffect associated with MAO-A inhibitors.

One of the metabolites of selegiline is its N-desmethyl analog.Structurally, the desmethylselegiline metabolite is the R(−)enantiomeric form of a secondary amine of the formula:

Heretofore, desmethylselegiline was not known to have pharmaceuticallyuseful MAO-related effects, i.e., potent and selective inhibitoryeffects on MAO-B. In the course of determining the usefulness ofdesmethylselegiline for the purposes of the present invention, theMAO-related effects of desmethylselegiline were more completelycharacterized. This characterization has established thatdesmethylselegiline has exceedingly weak MAO-B inhibitory effects and noadvantages in selectivity with respect to MAO-B compared to selegiline.

For example, the present characterization established that selegilinehas an IC₅₀ value against MAO-B in human platelets of 5×10⁻⁹ M whereasR(−)desmethylselegiline's IC₅₀ value is 4×10⁻⁷ M, indicating the latteris approximately 80 times less potent as an MAO-B inhibitor than theformer. Similar characteristics can be seen in the following datameasuring inhibition of MAO-B and MAO-A in rat cortex mitochondrial-richfractions:

TABLE 1 Inhibition of MAO by Selegiline and Desmethylselegiline PercentInhibition selegiline R(−)desmethylselegiline Conc. MAO-B MAO-A MAO-BMAO-A  0.003 μM 16.70 — 3.40 —  0.010 μM 40.20 — 7.50 —  0.030 μM 64.70— 4.60 —  0.100 μM 91.80 — 6.70 —  0.300 μM 94.55 9.75 26.15 0.0  1.000μM 95.65 32.55 54.73 0.70  3.000 μM 98.10 65.50 86.27 4.10  10.000 μM —97.75 95.15 11.75  30.000 μM — — 97.05 — 100.000 μM — — — 56.10

As is apparent from the above table, selegiline is approximately 128times more potent as an inhibitor of MAO-B relative to MAO-A, whereasdesmethylselegiline is about 97 times more potent as an inhibitor ofMAO-B relative to MAO-A. Accordingly, desmethylselegiline appears tohave an approximately equal selectivity for MAO-B compared to MAO-A asselegiline, albeit with a substantially reduced potency.

Analogous results are obtained in rat brain tissue. Selegiline exhibitsan IC₅₀ for MAO-B of 0.11×10⁻⁷ M whereas desmethylselegiline's IC₅₀value is 7.3×10⁻⁷ M, indicating desmethylselegiline is approximately 70times less potent as an MAO-B inhibitor than selegiline. Both compoundsexhibit low potency in inhibiting MAO-A in rat brain tissue, 0.18×10⁻⁵for selegiline, 7.0×10⁻⁵ for desmethylselegiline. Thus, in vitroR(−)desmethylselegiline is approximately 39 times less potent thanselegiline in inhibiting MAO-A.

Based on its pharmacological profile as set forth above,R(−)desmethylselegiline as an MAO-B inhibitor provides no advantages ineither potency or selectivity compared to selegiline. To the contrary,the above in vitro data suggest that use of desmethylselegiline as anMAO-B inhibitor requires on the order of 70 times the amount ofselegiline.

The potency of R(−)desmethylselegiline as an MAO-B inhibitor in vivo hasbeen reported by Heinonen, E. H., et al. (“Desmethylselegiline, ametabolite of selegiline, is an irreversible inhibitor of MAO-B in humansubjects,” referenced in Academic Dissertation “Selegiline in theTreatment of Parkinson's Disease,” from Research Reports from theDepartment of Neurology, University of Turku, Turku, Finland, No. 33(1995), pp. 59-61). According to Heinonen, desmethylselegiline in vivohas only about one-fifth the MAO-B inhibitory effect as selegiline,i.e., a dose of 10 mg of desmethylselegiline would be required for thesame MAO-B effect as 1.8 mg of selegiline. In rats, Barbe reportedR(−)desmethylselegiline to be an irreversible inhibitor of MAO-B, with apotency about 60 fold lower than selegiline in vitro and about 3 foldlower ex vivo (Barbe, H. O., J. Neural Trans.(Suppl.):32:131 (1990)).

The various diseases and conditions for which selegiline is discloasedas being useful include: depression (U.S. Pat. No. 4,861,800);Alzheimer's disease and Parkinson's disease, particularly through theuse of transdermal dosage forms, including ointments, creams andpatches; macular degeneration (U.S. Pat. No. 5,242,950); age-dependentdegeneracies, including renal function and cognitive function asevidenced by spatial learning ability (U.S. Pat. No. 5,151,449);pituitary-dependent Cushing's disease in humans and nonhumans (U.S. Pat.No. 5,192,808); immune system dysfunction in both humans (U.S. Pat. No.5,387,615) and animals (U.S. Pat. No. 5,276,057); age-dependent weightloss in mammals (U.S. Pat. No. 5,225,446); and schizophrenia (U.S. Pat.No. 5,151,419). PCT Published Application WO 92/17169 discloses the useof selegiline in the treatment of neuromuscular and neurodegenerativedisease and in the treatment of CNS injury due to hypoxia, hypoglycemia,ischemic stroke, or trauma. In addition, the biochemical effects ofselegiline on neuronal cells have been extensively studied. For example,see Tatton, et al., “Selegiline Can Mediate Neuronal Rescue Rather thanNeuronal Protection,” Movement Disorders 8 (Supp. 1):S20-S30 (1993);Tatton, et al., “Rescue of Dying Neurons,” J. Neurosci. Res. 30:666-672(1991); and Tatton, et al., “(−)-Deprenyl Prevents MitochondrialDepolarization and Reduces Cell Death in Trophically-Deprived Cells,”11th Int'l Symp. on Parkinson's Disease, Rome, Italy, Mar. 26-30, 1994.

Although selegiline is reported as being effective in treating theforegoing conditions, neither the precise number or nature of itsmechanism or mechanisms of action are known. However, there is evidencethat selegiline provides neuroprotection or neuronal rescue, possibly byreducing oxidative neuronal damage, increasing the amount of the enzymesuperoxide dismutase, and/or reducing dopamine catabolism. For example,PCT Published Application WO 92/17169 reports that selegiline acts bydirectly maintaining, preventing loss of, and/or assisting in, the nervefunction of animals.

Selegiline is known to be useful when administered to a subject througha wide variety of routes of administration and dosage forms. For exampleU.S. Pat. No. 4,812,481 (Degussa AG) discloses the use of concomitantselegiline-amantadine in oral, peroral, enteral, pulmonary, rectal,nasal, vaginal, lingual, intravenous, intraarterial, intracardial,intramuscular, intraperitoneal, intracutaneous, and subcutaneousformulations. U.S. Pat. No. 5,192,550 (Alza Corporation) describes adosage form comprising an outer wall impermeable to selegilire butpermeable to external fluids. This dosage form may have applicabilityfor the oral, sublingual or buccal administration of selegiline.Similarly, U.S. Pat. No. 5,387,615 discloses a variety of selegilinecompositions, including tablets, pills, capsules, powders, aerosols,suppositories, skin patches, parenterals, and oral liquids, includingoil-aqueous suspensions, solutions, and emulsions. Also disclosed areselegiline-containing sustained release (long acting) formulations anddevices.

Although a highly potent and selective MAO-B inhibitor, selegiline'spractical use is circumscribed by its dose-dependent specificity forMAO-B, and the pharmacology of selegiline metabolites generated afteradministration.

SUMMARY OF THE INVENTION

The present invention is based upon the surprising discovery that bothdesmethylselegiline (“DMS” or “R(−)DMS”) and its enantiomer(ent-desmethylselegiline, abbreviated as “ent-DMS” or “S(+)DMS”) areuseful in providing selegiline-like effects in subjects, notwithstandingdramatically reduced MAO-B inhibitory activity and an apparent lack ofenhanced selectivity for MAO-B compared to selegiline. It has beendiscovered that desmethylselegiline, ent-desmethylselegiline and theirisomeric mixtures provide a more advantageous way of obtainingselegiline-like therapeutic effects in selegiline-responsive diseases orconditions. This is particularly true for diseases or conditionscharacterized by neuronal degeneration, neuronal trauma or which arehypodopaminergic in nature, i.e. diseases or conditions characterized byreduced dopamine release and formation

Thus, the present invention provides novel pharmaceutical compositionsin which desmethylselegiline, either alone or in a racemic mixture withent-desmethylselegiline, is employed as the active ingredient and noveltherapeutic methods involving the administration of desmethylselegiline.Specifically, the present invention provides:

(1) An improved method for obtaining selegiline-like therapeutic effectsin a subject suffering from a selegiline-responsive disease orcondition, which comprises: administering to said subjectdesmethylselegiline in a dosage regime effective to produce saidselegiline-like therapeutic effect.

(2) A non-oral pharmaceutical composition comprising an amount ofdesmethylselegiline such that one or more unit doses of said compositionadministered on a periodic basis is effective to treat one or moreselegiline-responsive diseases or conditions in a subject to whom saidunit dose or unit doses are administered.

The pharmaceutical composition may contain desmethylselegiline in asubstantially enantiomerically pure state or, alternatively, thecomposition may contain a racemic mixture of enantiomers that aretogether present in an amount sufficient for one or more unit doses ofthe composition to be effective in treating a selegiline-responsivedisease or condition. The composition may be designed in such a way asto make it suitable for sublingual, buccal, parenteral or transdermaladministration and may be adapted for effecting neuronal rescue orprotection. The composition may also be adapted for restoring orimproving immune system function in a human.

In addition, the present invention is directed to a therapeutic packagefor dispensing to, or for use in dispensing to, a patient being treatedfor a neuronal-protective or neuronal-regenerative selegeline-responsivedisease or condition. The package contains one or more unit doses, eachsuch unit dose comprising an amount of desmethylselegiline such thatperiodic administration is effective in treating the patient'sselegeline-responsive disease or condition. The therapeutic package alsocomprises a finished pharmaceutical container containing the unit dosesof desmethylselegiline and further containing or comprising labelingdirecting the use of the package in the treatment of theselegiline-responsive disease or condition. The unit doses may beadapted for oral administration, e.g. as tablets or capsules, or may beadapted for non-oral administration.

The invention is also directed to a method of dispensingdesmethylselegiline to a patient being treated for a neuronal-protectiveor neuronal-regenerative selegeline-responsive disease or condition. Themethod comprises providing patients with a therapeutic package havingone or more unit doses of desmethylselegiline in an amount such thatperiodic administration to the patient is effective in treating theirselegeline-responsive disease or condition. The package also comprises afinished pharmaceutical container containing the desmethylselegiline andhaving labeling directing the use of the package in the treatment theselegeline-responsive disease or condition. The unit doses in thepackage may be adapted for either oral or non-oral administration.

As used herein the term “selegiline-responsive disease or condition”refers to any of the various diseases or conditions in mammals,including humans, for which selegiline is disclosed in the prior art asbeing useful. In particular, a “selegiline-responsive disease orcondition” refers to the various diseases and conditions describedabove, e.g., Alzheimer's disease, cognitive dysfunction, neuronalrescue, and the like. The term also refers to the use of selegiline asan appetite suppressant. Similarly, the term “selegiline-liketherapeutic effect” refers to one or more of the salutary effectsreported as being exerted by selegiline in subjects being treated for aselegiline-responsive disease or condition.

The selegiline-responsive diseases or conditions related to neuronaldegeneration or trauma which respond to the present methods includeParkinson's disease, Alzheimer's disease, depression, glaucoma, maculardegeneration, ischemia, diabetic neuropathy, attention deficit disorder,post polio syndrome, multiple sclerosis, impotence, narcolepsy, chronicfatigue syndrome, alopecia, senile dementia, hypoxia, cognitivedysfunction, negative symptomatology of schizophrenia, amyotrophiclateral sclerosis, Tourette's syndrome, tardive dyskinesia, and toxicneurodegeneration.

The present invention also encompasses the restoration or improvement ofimmune system function by R(−)DMS or mixtures of R(−)DMS and S(+)DMS.Such improvement or restoration has been reported to occur whenselegiline is administered to animals. The conditions or diseasestreatable include age-dependent immune system dysfunction, AIDS,infectious diseases and immunological loss due to cancer chemotherapy.

Desmethylselegiline may be administered either by a route involvinggastrointestinal absorption or by a route that does not rely upongastrointestinal absorption. Depending upon the particular routeemployed, desmethylselegiline is administered in the form of the freebase or as a physiologically acceptable non-toxic acid addition salt.Such salts include those derived from organic and inorganic acids suchas, without limitation, hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid, methanesulphonic acid, acetic acid, tartaric acid,lactic acid, succinic acid, citric acid, malic acid, maleic acid, sorbicacid, aconitic acid, salicylic acid, phthalic acid, embonic acid,enanthic acid, and the like. The use of salts, especially thehydrochloride, is particularly desirable when the route ofadministration employs aqueous solutions, as for example parenteraladministration; use of delivered desmethylselegiline in the form of thefree base is especially useful for transdermal administration.Accordingly, reference herein to the administration of DMS or ent-DMS orto mixtures thereof encompasses both the free base and acid additionsalt forms.

The optimal daily dose of desmethylselegiline, or of a combination ofR(−)DMS and S(+)DMS, useful for the purposes of the present invention isdetermined by methods known in the art, e.g., based on the severity ofthe disease or condition being treated, the condition of the subject towhom treatment is being given, the desired degree of therapeuticresponse, and the concomitant therapies being administered to thepatient or animal. Ordinarily, however, the attending physician orveterinarian will administer an initial dose of at least about 0.015mg/kg, calculated on the basis of the free secondary amine, withprogressively higher doses being employed depending upon the route ofadministration and the subsequent response to the therapy. Typically thedaily dose will be about 0.5 mg/kg and may extend to about 1.0 mg/kg ofthe patient's body weight depending on the route of administration.Again, all such doses should be calculated on the basis of the freesecondary amine. These guidelines further require that the actual dosebe carefully titrated by the attending physician or veterinariandepending on the age, weight, clinical condition, and observed responseof the individual patient or animal.

The daily dose can be administered in a single or multiple dosageregimen. Either oral or non-oral dosage forms may be used and maypermit, for example, a continuous release of relatively small amounts ofthe active ingredient from a single dosage unit, such as a transdermalpatch, over the course of one or more days. This is particularlydesirable in the treatment of chronic conditions such as Parkinson'sdisease, Alzheimer's disease, and depression. Alternatively, it can bedesirable in conditions such as ischemia or neural damage to administerone or more discrete doses by a more direct systemic route such asintravenously or by inhalation. In still other instances such asglaucoma and macular degeneration, localized administration, such as viathe intraocular route, can be indicated.

Pharmaceutical compositions containing desmethylselegiline and/orent-desmethylselegiline can be prepared according to conventionaltechniques. For example, preparations for parenteral routes ofadministration for desmethylselegiline, e.g., intramuscular, intravenousand intraarterial routes, can employ sterile isotonic saline solutions.Sterile buffered solutions can also be employed for intraocularadministration.

Transdermal dosage unit forms of desmethylselegiline and/orent-desmethylselegiline can be prepared utilizing a variety ofpreviously described techniques (see e.g., U.S. Pat. Nos. 4,861,800;4,868,218; 5,128,145; 5,190,763; and 5,242,950; and EP-A 404807, EP-A509761, and EP-A 593807). For example, a monolithic patch structure canbe utilized in which desmethylselegiline is directly incorporated intothe adhesive and this mixture is cast onto a backing sheet.

Alternatively desmethylselegiline, and/or ent-desmethylselegiline, canbe incorporated as an acid addition salt into a multilayer patch whicheffects a conversion of the salt to the free base, as described forexample in EP-A 593807.

Desmethylselegiline and/or ent-desmethylselegiline can also beadministered by a device employing a lyotropic liquid crystallinecomposition in which, for example, 5 to 15% of desmethylselegiline iscombined with a mixture of liquid and solid polyethylene glycols, apolymer, and a nonionic surfactant, optionally with the addition ofpropylene glycol and an emulsifying agent. For further details on thepreparation of such transdermal preparations, reference can be made toEP-A 5509761.

Since the term “ent-desmethylselegiline” refers to the S(+) isomericform of desmethylselegiline, reference above to mixtures ofdesmethylselegiline and ent-desmethylselegiline includes both racemicand non-racemic mixtures of optical isomers.

Subjects treatable by the present preparations and methods include bothhuman and non-human subjects for which selegiline-like therapeuticeffects are known to be useful. Accordingly, the compositions andmethods above provide especially useful therapies for mammals,especially domesticated mammals. Thus, the present methods andcompositions are used in treating selegiline-responsive diseases orconditions in canine and feline species.

Successful use of the compositions and methods above requires employmentof an effective amount of desmethylselegiline, or mixtures ofdesmethylselegiline and ent-desmethylselegiline. Although bothdesmethylselegiline and ent-desmethylselegiline are dramatically lesspotent than selegiline as inhibitors of MAO, employment of these agents,or a mixture of these agents, for neuroprotection does not require acommensurately increased dosage to obtain a selegiline-like therapeuticresponse. Surprisingly, dosages necessary to attain a selegiline-liketherapeutic appear to be on the same order as the known doses ofselegiline. Accordingly, because both desmethylselegiline andent-desmethylselegiline exhibit a much lower inhibition of MAO-A at suchdosages, desmethylselegiline and ent-desmethylselegiline provide asubstantially wider margin of safety with respect to MAO-A associatedtoxicity compared to selegiline. In particular, the risk of the adverseeffects of MAO-A inhibition, e.g., hypertensive crisis, are minimizeddue to the 40-70 fold reduced potency for MAO-A inhibition.

As described above and notwithstanding its demonstrably inferiorinhibitory properties with respect to MAO-B inhibition,desmethylselegiline and its enantiomer appear to be at least aseffective as selegiline in treating certain selegiline-responsiveconditions, e.g., conditions resulting from neuronal degeneration orneuronal trauma. Although the oral route of administration willgenerally be most convenient, drug may be administered by theparenteral, topical, transdermal, intraocular, buccal, sublingual,intranasal, inhalation, vaginal, rectal or other routes as well.

As noted above, the present invention encompasses the additionaldiscovery that desmethylselegiline can be employed either alone or inmixture with desmethylselegiline. Desmethylselegiline, its enantiomerand mixtures thereof are conveniently prepared by methods known in theart, as described in Example 1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: HPLC Chromatogram of Purified R(−)DMS (Microsorb MV CyanoColumn). The purity of a preparation of R(−)DMS was determined by HPLCon a Microsorb MV Cyano column and results are shown in FIG. 1. Thecolumn had dimensions of 4.6 mm×15 cm and was developed at a flow rateof 1.0 ml/min using a mobile phase containing 90% 0.01 M H₃PO₄(pH 3.5)and 10% acetonitrile. The column was run at a temperature of 40° C. andeffluent was monitored at a wavelength of 215 nm. The chromatogram showsone major peak appearing at a time of 6.08 minutes and having 99.5% ofthe total light-absorbing material eluted from the column. No other peakhad greater than 0.24%.

FIG. 2: HPLC Elution Profile of R(−)DMS (Zorbax Mac-Mod C18 Column). Thesame preparation that was analyzed in the experiments discussed in FIG.1 was also analyzed for purity by HPLC on a Zorbax Mac-Mod SB-C18 column(4.6 mm×75 mm). Effluent was monitored at 215 nm and results can be seenin FIG. 2. Greater than 99.6% of the light-absorbing material appearedin the single large peak eluting at a time of between 2 and 3 minutes.

FIG. 3: Mass Spectrum of R(−)DMS. A mass spectrum was obtained forpurified R(−)DMS and results are shown in FIG. 3. The spectrum isconsistent with a molecule having a molecular weight of 209.72 and amolecular formula of C₁₂H₁₅N.HCl.

FIG. 4: Infrared Spectrum (KBr) of Purified R(−)DMS. Infraredspectroscopy was performed on a preparation of R(−)DMS and results areshown in FIG. 4. The solvent used was CDCl₃.

FIG. 5: NMR Spectrum of Purified R(−)DMS. A preparation Purified R(−)DMSwas dissolved in CDCl₃ and ¹H NMR spectroscopy was performed at 300 MHZ.Results are shown in FIG. 5.

FIG. 6: HPLC Chromatogram of S(+)DMS. The purity of a preparation ofS(+)DMS was examined by reverse phase HPLC on a 4.6 mm×75 mm ZorbaxMac-Mod SB-C18 column. The elution profile, monitored at 215 nm, isshown in FIG. 6. One major peak appears in the profile at a time ofabout 3 minutes and contains greater than 99% of the totallight-absorbing material that eluted from the column.

FIG. 7: Mass Spectrum of Purified S(+)DMS. Mass spectroscopy wasperformed on the same preparation examined in FIG. 6. The spectrum isshown in FIG. 7 and is consistent with the structure of S(+)DMS.

FIG. 8: Infrared Spectrum (KBr) of Purified S(+)DMS. The preparation ofS(+)DMS discussed in connection with FIGS. 6 and 7 was examined byinfrared spectroscopy and results are shown in FIG. 8.

FIG. 9: Effect of Selegiline on Neuron Survival. Mesencephalic cultureswere prepared from embryonic 14 day rats. Cultures were used at about1.5 million cells per plate and were maintained either in growth mediumalone (control cultures) or in growth medium supplemented withselegiline. On day 1, 8 and 15, cells were immunostained for thepresence of tyrosine hydroxylase (“TH”). Striped bars represent resultsobtained for cultures maintained in the presence of 50 μM selegiline andopen bars represent results for control cultures. In all cases, resultsare expressed as a percentage of TH positive cells present in controlcultures on day 1. The abbreviation “DIV” refers to “days in vitro.”Asterisks or stars above bars both in FIG. 9 and the figures discussedbelow indicates a result that differs from controls in an amount that isstatistically significant, i.e. P<0.05.

FIG. 10: [³H]-Dopamine Uptake in Mesencephalic Cells. Cells, cultured asdescribed above for FIG. 9, were tested for their uptake of labeleddopamine and results are shown in FIG. 10. Striped bars represent uptakein cells maintained in the presence of 50 μM selegiline and open barsrepresent uptake in control cultures.

FIG. 11: Effect of Selegiline on Glutamate Receptor Dependent NeuronalCell Death. Rat embryonic mesencephalic cells were cultured as describedabove. After allowing cultures to stabilize, the culture medium waschanged daily for a period of 4 days to induce glutamatereceptor-dependent cell death. Depending on the culture, mediumcontained either 0.5, 5.0 or 50 μM selegiline. After the final mediumchange, cultured cells were immunostained for the presence of tyrosinehydroxylase. From left to right, bars represent results for controls,0.5, 5.0 and 50 μM selegiline.

FIG. 12: Effect of Selegiline on Dopamine Uptake in Neuronal Cultures.Rat mesencephalic cells were cultured and medium was changed on a dailybasis as discussed for FIG. 11. Uptake of tritiated dopamine by cellswas measured and results are shown in the figure. From left to right,bars are in the same order as for FIG. 11.

FIG. 13: Effect of R(−)Desmethylselegiline on Glutamate ReceptorDependent Neuronal Cell Death. Rat embryonic mesencephalic cultures wereprepared as described above except that R(−)DMS was used instead ofselegiline. On day 9, the number of TH positive cells in cultures wasdetermined. Results are expressed as a percentage of control. From leftto right, bars show results for controls, 0.5, 5 and 50 μM R(−)DMS.

FIG. 14: Effect of R(−)Desmethylselegiline on Dopamine Uptake inNeuronal Cultures. Cell cultures were prepared as described above forFIG. 13 and then tested for uptake of tritiated dopamine. Results forcontrols and for cells maintained in the presence of 0.5 μM, 5 μM and 50μM desmethylselegiline are shown from left to right in the figure.

FIG. 15: Comparison of Dopamine Uptake in Mesencephalic Cells Incubatedin the Presence of Different Monoamine Oxidase Inhibitors. Rat embryonicmesencephalic cells were prepared as described for FIGS. 11-14 andincubated in the presence of a variety of monoamine oxidase inhibitors.The inhibitors examined were selegiline; R(−) desmethylselegiline;pargyline; and clorgyline, all at concentrations of 0.5, 5 and 50 μM. Inaddition, cells were incubated in the presence of the glutamate receptorblocker MK-801 at a concentration of 10 μM. Cultures were tested foruptake of tritiated dopamine.

FIG. 16: Relative Effectiveness of R(−) and S(+)DMS in Maintaining[³H]-Dopamine Uptake by Cultured Mesencephalic Cells (NMDA Model).Preparations of R(−) and S(+)DMS were assayed for their effect on[³H]-dopamine uptake by cultured rat mesencephalic cells exposed to thetoxin N-methyl-D-aspartate (NMDA). Results were expressed as apercentage of the uptake seen in control cultures not exposed to NMDAand are shown in FIG. 16. From the left, the bars represent: cellsincubated with medium alone; medium+5 μM deprenyl; medium+0.5 μMR(−)DMS; medium+5 μM R(−)DMS; medium+50 μM R(−)DMS; medium+0.5 μMS(+)DMS; medium+5 μM S(+)DMS; and medium+50 μM S(+)DMS. Al of the cellcultures shown in the figure were exposed to 100 μM NMDA. Statisticalsignificance was determined by ANOVA followed by Dunnett's test. Onestar above a bar indicates a percentage uptake that differssignificantly from control uptake at the 0.05 confidence level. Twostars indicate a result that differs at the 0.01 confidence level.

FIG. 17: Relative Effectiveness of R(−) and S(+)DMS on Survival ofCultured Mesencephalic Cells (NMDA Model). Rat mesencephalic cellcultures were exposed to 100 μM NMDA and incubated as described above inconnection with FIG. 16. The effect of DMS enantiomers on the survivalTH positive cells is shown in FIG. 17. The bars are in the same order asfor FIG. 16 and results are expressed as a percentage of control. Onestar indicates p<0.05 and two stars indicates p<0.01 when results arecompared to those obtained for cells exposed to NMDA and then incubatedin unsupplemented medium.

FIG. 18: Inhibition of Neuronal Dopamine Re-Uptake by Deprenyl and theTwo Enantiomers of Desmethylselegiline. An in vitro nerve terminalpreparation (synaptosome preparation) was prepared using fresh ratneostriatal tissue. This was examined for its ability to take uptritiated dopamine in buffer alone or in buffer supplemented withvarious concentrations of selegiline, R(−)desmethylselegiline orS(+)desmethylselegiline. Uptake in the presence of each MAO inhibitor,expressed as a percent inhibition vs. log concentration of inhibitor isshown in FIG. 18. As indicated, the plot was used to determine the IC₅₀for each test agent.

FIG. 19: Determination of IC₅₀ Values for Inhibition of DopamineRe-Uptake. The experiment of FIG. 18 was repeated in a concentrationrange designed to more accurately provide an IC₅₀ value and results areshown in FIG. 19. Using the log C vs. probit graphs, as shown in thefigure, the IC₅₀ for S(+)DMS was determined to be about 11 μM; forselegiline, about 46 μM; and for R(−)DMS about 54 μM.

FIG. 20: In Vivo MAO-B Inhibition in Guinea Pig Hippocampus. Variousdoses of selegiline, R(−)desmethylselegiline, andS(+)desmethylselegiline were administered daily into guinea pigs for aperiod of 5 days. Animals were then sacrificed and the MAO-B activity inthe hippocampus portion of the brain was determined. Results wereexpressed as a percent inhibition relative to hippocampus MAOB activityin control animals and are shown in FIG. 20. The plots were used toestimate the ID₅₀ dosage for each agent. The ID₅₀ for selegiline wasabout 0.008 mg/kg; and for R(−)DMS, it was about 0.2 mg/kg; and forS(+)DMS, it was about 0.5 mg/kg.

DETAILED DESCRIPTION OF THE INVENTION

The surprising utility of desmethylselegiline andent-desmethylselegiline in treating selegiline-responsive diseases orconditions is attributable in part to their powerful action inpreventing loss of dopaminergic neurons by promoting repair andrecovery. Because desmethylselegiline prevents loss and facilitatesrecovery of nerve cell function, it is of value in a wide variety ofneurodegenerative and neuromuscular diseases. In this regard,desmethylselegiline is at least as potent as selegiline, and in onemodel of neuroprotection, appeared to be substantially more potent. Thisis described more empirically in Examples 4 to 9 below.

EXAMPLES Example 1 Preparation of Desmethylselegiline andEnt-desmethylselegiline

A. Desmethylselegiline

Desmethylselegiline (designated below as “R(−)DMS”) is prepared bymethods known in the art. For example, desmethylselegiline is a knownchemical intermediate for the preparation of selegiline as described inU.S. Pat. No. 4,925,878. Desmethylselegiline can be prepared by treatinga solution of R(−)-2-aminophenylpropane (levoamphetamine):

in an inert organic solvent such as toluene with an equimolar amount ofa reactive propargyl halide such as propargyl bromide, Br—CH₂—C≡CH, atslightly elevated temperatures (70°-90° C.). Optionally the reaction canbe conducted in the presence of an acid acceptor such as potassiumcarbonate. The reaction mixture is then extracted with aqueous acid, forexample 5% hydrochloric acid, and the extracts are rendered alkaline.The nonaqueous layer which forms is separated, for example by extractionwith benzene, dried, and distilled under reduced pressure.

Alternatively the propargylation can be conducted in a two-phase systemof a water-immiscible solvent and aqueous alkali, utilizing a salt ofR(+)-2-aminophenylpropane with a weak acid such as the tartrate,analogously to the preparation of selegiline as described in U.S. Pat.No. 4,564,706.

B. Ent-Desmethylselegiline

Ent-desmethylselegiline (designated below as “S(+)DMS”) is convenientlyprepared from the enantiomeric S(+)-2-aminophenylpropane(dextroamphetamine), i.e.,

following the procedures set forth above for desmethylselegiline.

C. Mixtures of Enantiomers

Mixtures of enantiomeric forms of desmethylselegiline, including racemicdesmethylselegiline, are conveniently prepared from enantiomericmixtures, including racemic mixtures of the above aminophenylpropanestarting material.

D. Conversion Into Acid Addition Salts

N-(prop-2-ynyl)-2-aminophenylpropane in either optically active orracemic form can be converted to a physiologically acceptable non-toxicacid addition salt by conventional techniques such as treatment with amineral acid. For example, hydrogen chloride in isopropanol is employedin the preparation of desmethylselegiline hydrochloride. Either the freebase or salt can be further purified, again by conventional techniquessuch as recrystallization or chromatography.

Example 2 Characteristics of Substantially Pure R(−)DMS

A preparation of substantially pure R(−)DMS has the appearance of awhite crystalline solid with a melting point of 162-163° C. and anoptical rotation of [α]_(D) ²³° C.=−15.2+/−2.0 when measured at aconcentration of 1.0 M using water as solvent. R(−)DMS appeared to be99.5% pure when analyzed by HPLC on a Microsorb MV Cyano column (seechromatogram in FIG. 1) and 99.6% pure when analyzed by HPLC on a ZorbaxMac-Mod SB-C18 column (see chromatogram in FIG. 2). No single impurityis present at a concentration greater than or equal to 0.5%. Heavymetals are present at a concentration of less than 10 ppm andamphetamine hydrochloride at a concentration of less than 0.03%. Thelast solvents used for dissolving the preparation, ethyl acetate andethanol are both present at a concentration of less than 0.1%. A massspectrum performed on the preparation (see FIG. 3) is consistent with acompound having a molecular weight of 209.72 and a formula ofC₁₂H₁₅N.HCl. Infrared and NMR spectra are shown in FIGS. 4 and 5respectively. These are also consistent with the known structure ofR-(−)-DMS.

Example 3 Characteristics of Substantially Pure S(+)DMS

A preparation of substantially pure S(+)DMS has the appearance of awhite powder with a melting point of approximately 160.04° C. and aspecific rotation of +15.1 degrees when measured at 22° C. in water, ata concentration of 1.0 M. When examined by reverse phase HPLC on aZorbax Mac-Mod SB-C18 column the preparation appears to be about 99.9%pure (FIG. 6). Amphetamine hydrochloride is present at a concentrationof less than 0.13% (w/w). A mass spectrum is performed on thepreparation and is consistent with a compound having a molecular weightof 209.72 and a molecular formula of C₁₂H₁₅N.HCl (see FIG. 7). Infraredspectroscopy is performed and also provides results consistent with thestructure of S(+)DMS (see FIG. 8).

Example 4 Neuronal Survival as Measured Using Tyrosine Hydroxylase

The effect of desmethylselegiline on neuron survival can be correlatedto tyrosine hydroxylase, the rate limiting enzyme in dopaminebiosynthesis. Assays are performed by determining the number of tyrosinehydroxylase positive cells in cultured E-14 embryonic mesencephaliccells over a period of 7 to 14 days. Protection in this system has beenseen with a variety of trophic factors including BDNF, GDNF, EGF, andβ-FGF.

A. Test Methods

Timed pregnant Sprague-Dawley rats are used to establish neuronalcultures from embryonic rat brain on the 14th day of gestation.Mesencephalon is dissected out without the membrane coverings andcollected in Ca⁺⁺ and Mg⁺⁺ free balanced salt solution at 4° C. Tissuefragments are dissociated in chemically defined medium by mildtrituration with a small bore pasteur pipette. Cell suspension is platedin polyornithine-coated 35 mm Falcon plastic dishes (0.1 mg/ml, Sigma)at a density of 1.5×10⁶ cells/dish. Cultures are maintained at 37° C. inan atmosphere of 10% CO₂/90% air and 100% relative humidity, and fedtwice weekly with chemically defined medium consisting of MEM/F12 (1:1,Gibco), glucose (33 mM), HEPES (15 mM), NaHCO₃ (44.6 mM), transferrin(100 mg/ml), insulin (25 mg/ml), putrescine (60 nM), sodium selenite (30nM), progesterone (20 nM), and glutamine (2 mM). Control cells receiveno further additions. The medium used for other cells also included testsubstance, e.g. selegiline, at one or more concentrations.

Cultures are fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH7.4) for 30 minutes at room temperature, permeabilized with 0.2% TritonX-100 for 30 minutes and incubated with an antibody against tyrosinehydroxylase (1:1000; Eugene Tech) for 48 hours at 4° C. in the presenceof a blocking serum. They are then stained using a peroxidase-coupledavidin-biotin staining kit (Vectastain ABC kit; Vector Labs) with3′,3′-diaminobenzidine as a chromagen.

The number of dopaminergic neurons in cultures is determined by countingthe cells positively immunostained with TH antibodies. 100 fields (0.5mm×0.5 mm) in two transverse strips across the diameter of the dish,representing 2.5% of the total area, are counted using a Nikon invertedmicroscope at 200× magnification.

B. Results

Using the procedures described above, the following results wereobtained:

TABLE 2 Effect of Selegiline and DMS on the Survival of TH PositiveCells Control Selegiline Desmethylselegiline Mean Mean Mean Conc.cells/cm² cells/cm² % cont. cells/cm² % cont. 0.5 μM 108.55 201.70 ±25.01 185.81 246.00 ± 22.76 226.62   5 μM — 237.00 ± 12.59 218.33 357.95± 25.76 329.76  50 μM — 292.28 ± 17.41 269.25 391.60 ± 34.93 360.76

Example 5 Neuronal Survival as Measured Using Dopamine Uptake

In addition to determining the number of TH positive cells in culture(see Example 4) the protective effect of desmethylselegiline on neuronalcells also can be determined by directly measuring dopamine uptake. Theamount of uptake by the cultured brain cells corresponds to axonalgrowth.

A. Test Methods

Cell cultures, established in the manner discussed above, are incubatedwith [³H]dopamine (0.5 mCi/ml; 37 Ci/mmol; New England Nuclear) for 15minutes in the presence of ascorbic acid (0.2 mg/ml) in PBS (pH 7.3),supplemented with 0.9 mM CaCl₂ and 0.5 mM MgCl₂ at 37° C. After tworinses and a 5 minute incubation with fresh buffer, [³H]dopamineaccumulated within the cells is released by incubating the cultures with95% ethanol for 30 minutes at 37° C. Preparations are then added to 10ml Ecoscint (National Diagnostics) and counted in a scintillationspectrometer. Nonspecific uptake values are obtained by blockingdopaminergic neuronal uptake with 10 mM mazindol.

B. Results

Using the above procedure, the results shown in Table 3 were obtained.

TABLE 3 Effect of Selegiline and DMS on ³H-Dopamine Uptake Cont.Selegiline Desmethylselegiline Conc. Mean Mean % Cont Mean % Cont 0.5 μM11982 14452 ± 212  120.6 24020 ± 800  200.4   5 μM — 16468 ± 576  137.534936 ± 2119 291.5  50 μM — 33018 ± 1317 275.5 56826 ± 2656 474.3

C. Conclusions from Examples 4 and 5

The results described in Examples 4 and 5 indicate thatdesmethylselegiline is substantially more potent superior to selegilineas a neuroprotective agent. This is true notwithstanding the fact thatdesmethylselegiline in much less potent than selegiline as an inhibitorof MAO-B.

Example 6 Neuroprotective Action of Desmethylselegiline Enantiomers inCultured Dopamine-Containing Mesencephalic Neurons In Vitro

The survival of mesencephalic, dopamine-containing neuronal cultures ofrat brain tissue was used in these experiments to examineneuroprotective properties of selegiline and R(−) desmethylselegiline.The number of TH positive neurons is directly proportional to thesurvival of dopaminergic neurons and ³H-dopamine uptake is a measure ofaxonal growth in these neurons

A. Effect of Selegiline on the Survival of Dopaminergic Neurons.

Mesencephalic cultures prepared from embryonic day 14 rats were treatedwith 0.5, 5 or 50 μM selegiline for 15 days, beginning on the day ofplating. (For a more detailed discussion of the culturing of cells andother methods used in these experiments see Mytilineou et al., J.Neurochem.61:1470-1478 (1993)). Survival and growth of dopamine neuronswas evaluated by tyrosine hydroxylase (TH) immunocytochemistry and[³H]dopamine uptake and results are shown in FIGS. 9 and 10.

B. Effect of Selegiline on Glutamate Receptor Dependent Cell Death.

The neuroprotective effect of selegiline was also examined using anexperimental paradigm that causes neuronal cell death that can beblocked by inhibition of glutamate receptors. In these experiments,cells were plated and allowed to stabilize for several days. The growthmedium of the cells was then changed on a daily basis to induce celldeath that can be prevented by blocking glutamate receptors, e.g. usingMK-801. After 4 days of daily medium changes, cultures were stained fortyrosine hydroxylase and assayed for uptake of tritiated dopamine. Theresults shown in FIGS. 11 and 12 further support the conclusion thatselegiline promotes the survival of dopaminergic neurons.

C. Effect of Desmethylselegiline on the Survival of Dopamine Neurons.

Using the glutamate receptor dependent model of neuron death, an evenmore potent protection of dopaminergic neurons was provided whendesmethylselegiline was used in place of selegiline. Even at the lowestdose tested (0.5 μM), desmethylselegiline caused a significant reductionin the loss of TH positive neurons (FIG. 13) and a significant increasein dopamine uptake (FIG. 14) relative to control cultures in whichmedium was used without supplementation with either selegiline ordesmethylselegiline.

D. Comparison with other MAO Inhibitors.

Using the glutamate receptor dependent model of neurotoxicity, theeffects of selegiline and desmethylselegiline were compared with twoother MAO inhibitors, pargyline and clorgyline (FIG. 15). In agreementwith previous results, measurement of dopamine uptake indicated neuronprotection by 50 μM deprenyl and 5 and 50 μM desmethylselegiline.Pargyline did not appear to offer any protection at the concentrationsused, while clorgyline protected at 50 μM. As expected, protection wasalso obtained by the NMDA receptor blocker MK-801 (10 μM).

E. Effect of DMS Enantiomers on ³H-Dopamine Uptake

The data summarized in Table 4 suggests that both (R−)DMS and S(+)DMSare effective as neuroprotectants in mesencephalic dopamine-containingneurons in culture.

TABLE 4 Effect of DMS Enantiomers on Dopamine Uptake ³H-Dopamine uptakeTreatment as a percentage + SEM Control   100 ± 14.14% R(−)DMS (10 μM)140.82 ± 26.20% S(+)DMS (10 μM)   234 ± 38.36%

These results were obtained using the medium change model of cell death.Compared to untreated control cells, there was 40% and 134% more axonalgrowth and terminal axonal survival after treatment with R(−)DMS andS(+)DMS, respectively. In this study, S(+)DMS showed greater potency asa neuroprotectant than R(−)DMS.

Example 7 Comparison of the Neuroprotective Effect of R(−)DMS andS(+)DMS

The neuroprotective effect of R(−)DMS and S(+)DMS on cultured ratmesencephalic cells was examined using two models of neuronal celldeath. In the first model, cells were exposed to 100 μMN-methyl-D-aspartate (NMDA), an agent which causes cell death by bindingto glutamate receptors. Cells exposed to NMDA were incubated in thepresence of either medium alone; medium supplemented with 50 μMdeprenyl; medium with 0.5, 5, or 50 μM R(−)DMS; or medium containing0.5, 5 or 50 μM S(+)DMS. The effect of these treatments on [³H]-dopamineuptake and the survival of TH positive cells was determined and resultsare shown in Tables 5-8 and FIGS. 16 and 17. It can be seen that bothforms of DMS had a neuroprotective effect, with S(+)DMS being the mosteffective treatment to a statistically significant degree as determinedby tritiated dopamine uptake. Experiments examining the neuroprotectiveeffect of DMS enantiomers were also performed using the medium changemodel of cell death described previously (see Example 6). As can be seenin Tables 9-12, both the R(−) and S(+) enantiomers significantlyenhanced [³H]-dopamine uptake and the survival of TH positive cells. Inthis model, the relative potency of both enantiomers appears to be equalto treatment with 50 μM selegiline.

TABLE 5 R(−)DMS: Dopamine Uptake After 100 μM NMDA Exposure ControlR(−)DMS (0.5 μM) R(−)DMS (5.0 μM) R(−) DMS (50 μM) Deprenyl (50 μM)counts/min counts/min % control counts/min % control counts/min %control counts/min % control 6013 9385 138.9 13509 199.9 23090 341.818479 273.5 6558 8976 132.9 11471 169.8 21530 318.7 16958 251.0 74629028 133.6 13786 204.0 17520 259.3 17550 259.8 6432 8133 120.4 10229151.4 22963 339.9 18572 274.9 7317 11304 167.3 11014 163.0 17708 262.115410 228.1 Mean 6756.4 9365.2 138.6 12001.8 177.6 20562.2 304.3 17393.8257.4 St. Dev. 614.3 1177.2 17.4 1569.7 23.2 2761.0 40.9 1295.7 19.2

TABLE 6 S(+)DMS: Dopamine Uptake After 100 μM NMDA Exposure ControlS(+)DMS (0.5 μM) S(+)DMS (5.0 μM) S(+) DMS (50 μM) Deprenyl (50 μM)counts/min counts/min % control counts/min % control counts/min %control counts/min % control 6013 12092 179.0 20313 300.6 25944 384.018479 273.5 6558 12269 181.6 16579 245.4 28545 422.5 16958 251.0 746216399 242.7 15929 235.8 39042 577.9 17550 259.8 6432 11435 169.2 15052222.8 33024 488.8 18572 274.9 7317 11096 164.2 15535 229.9 25101 371.515410 228.1 Mean 6756.4 12658.2 187.4 16681.6 246.9 30331.2 448.917393.8 257.4 St. Dev. 614.3 2144.9 31.7 2105.6 31.2 5764.6 85.3 1295.719.2

TABLE 7 R(−)DMS: TH Immunochemistry After 100 μM NMDA Exposure ControlR(−)DMS (0.5 μM) R(−)DMS (5.0 μM) R(−) DMS (50 μM) Deprenyl (50 μM)cells/cm² cells/cm² % control cells/cm² % control cells/cm² % controlcells/cm² % control 95.0 95.0 100.9 142.5 151.3 237.5 252.2 230.0 244.290.0 75.0 79.6 122.5 130.1 170.0 180.5 287.5 305.3 97.5 105.0 111.5130.0 138.1 102.5 108.8 187.5 199.1 117.5 124.8 115.0 122.1 177.5 188.5Mean 94.17 91.67 97.3 128.13 136.1 156.25 165.9 220.63 234.3 St. Dev.3.8 15.3 16.2 10.9 11.5 61.6 65.4 50.1 53.2

TABLE 8 S(+)DMS: TH Immunochemistry After 100 μM NMDA Exposure ControlS(+)DMS (0.5 μM) S(+)DMS (5.0 μM) S(+) DMS (50 μM) Deprenyl (50 μM)cells/cm² cells/cm² % control cells/cm² % control cells/cm² % controlcells/cm² % control 95.0 127.5 135.4 192.5 204.4 297.5 315.9 230 244.290.0 210 223.0 187.5 199.1 202.5 215.0 287.5 305.3 97.5 177.5 188.5192.5 204.4 317.5 337.2 187.5 199.1 172.5 183.2 222.5 236.3 177.5 188.5Mean 94.17 171.67 182.3 186.25 197.8 260 276.1 220.63 234.3 St. Dev.41.6 44.1 9.5 10.1 56.1 59.5 50.1 53.2

TABLE 9 R(−)DMS: Dopamine Uptake, Medium Change Model Control R(−)DMS(0.5 μM) R(−)DMS (5.0 μM) R(−) DMS (50 μM) Deprenyl (50 μM) counts/mincounts/min % control counts/min % control counts/min % controlcounts/min % control 17880 19885 142.3 32577 155.2 37440 178.3 38053181.2 21500 32002 152.4 29831 142.1 39200 186.7 34130 162.6 23471 29934142.6 36370 173.2 39126 186.3 36810 175.3 21134 27382 130.4 30342 144.540013 190.6 33863 161.3 Mean 20996.25 29800.75 141.9 32280 153.738944.75 185.5 35714 170.1 St. Dev. 2317.2 1890.4 9.0 2976.0 14.2 1080.75.1 2050.0 9.8

TABLE 10 S(+)DMS: Dopamine Uptake, Medium Change Model Control S(+)DMS(0.5 μM) S(+)DMS (5.0 μM) S(+) DMS (50 μM) Deprenyl (50 μM) counts/mincounts/min % control counts/min % control counts/min % controlcounts/min % control 17880 35830 170.6 35976 171.3 26002 123.8 38053181.2 21500 32074 152.8 36476 173.7 37320 177.7 34130 162.6 23471 33042157.4 38143 181.7 30725 146.3 36810 175.3 21134 39516 188.2 40964 195.138020 181.1 33863 161.3 Mean 20996.25 35115.5 167.2 37889.75 180.533016.75 157.3 35714 170.1 St. Dev. 2317.2 3337.9 15.9 2249.2 10.75715.7 27.2 2050.0 9.8

TABLE 11 R(−)DMS: TH Immunochemistry, Medium Change Model ControlR(−)DMS (0.5 μM) R(−)DMS (5.0 μM) R(−) DMS (50 μM) Deprenyl (50 μM)cells/cm² cells/cm² % control cells/cm² % control cells/cm² % controlcells/cm² % control 270.0 340.0 129.0 322.5 122.3 310.0 117.6 385.0146.0 237.0 310.0 117.6 342.5 129.9 442.5 167.9 327.5 124.2 280.0 330.0125.2 362.5 137.5 380.0 144.1 320.0 121.4 267.5 362.5 365.0 138.5 395.0149.8 Mean 263.63 335.63 123.9 348.13 132.1 381.88 144.9 344.17 130.6St. Dev. 18.6 21.8 5.8 19.8 7.5 54.8 20.8 35.6 13.5

TABLE 12 S(+)DMS: TH Immunochemistry, Medium Change Model ControlS(+)DMS (0.5 μM) S(+)DMS (5.0 μM) S(+) DMS (50 μM) Deprenyl (50 μM)cells/cm² cells/cm² % control cells/cm² % control cells/cm² % controlcells/cm² % control 270.0 402.5 152.7 342.5 129.9 307.5 116.6 385.0146.0 237.0 330.0 125.2 357.5 135.6 250.0 94.8 327.5 124.2 280.0 402.5152.7 325.0 123.3 312.5 118.5 320.0 121.4 267.5 477.5 352.5 133.7 287.5109.1 Mean 263.6 403.1 143.5 344.4 130.6 289.4 109.8 344.2 130.6 St.Dev. 18.6 60.2 15.9 14.3 5.4 28.4 10.8 35.6 13.5

Example 8 Desmethylselegiline and Ent-Desmethylselegiline as Inhibitorsof Dopamine Re-Uptake

The biological actions of the brain neurotransmitter dopamine areterminated at the synapse by a high-affinity, sodium andenergy-dependent transport system (neuronal re-uptake) present withinthe limiting membrane of the presynaptic dopamine-containing nerveterminal. Inhibition of this transport mechanism would extend theactions of dopamine at the synapse and therefore enhance dopaminesynaptic transmission.

A. Method of Testing

The R(−) and S(+) enantiomers of desmethylselegiline (DMS) were testedfor their ability to inhibit the dopamine re-uptake system and comparedto selegiline. Inhibitory activity in this assay is indicative of agentsof value in the treatment of diseases which require enhanced synapticdopamine activity. Presently this would include Parkinson's disease,Alzheimer's disease and attention deficit hyperactivity disorder (ADHD).

The assay system used was essentially that described by Fang et at.(Neuro-pharmacology 33:763-768 (1994)). An in vitro nerve-terminalpreparation (synaptosome preparation) was obtained form fresh ratneostriatal brain tissue. Transport by dopamine nerve-terminals wasestimated by measuring the uptake of tritiated dopamine.

B. Results

As seen in the data presented in Table 13, selegiline, R(−)DMS andS(+)DMS all inhibited dopamine re-uptake by dopamine-containing nerveterminals. Selegiline and R(−) DMS were approximately equipotent. Incontrast, S(+)DMS was 4-5 times more potent than either selegiline orR(−)DMS.

TABLE 13 ³H-Dopamine Uptake By Rat Neostriatal Brain Tissue % ReductionAgent Concentration {overscore (x)} ± SEM Dopamine  1 μM 52.0 ± 4.9  10μM 80.9 ± 0.4 Selegiline 100 nM  7.0 ± 3.6  1 μM 13.9 ± 4.7  10 μM 16.3± 3.8 100 μM 59.8 ± 1.0 R(−)DMS 100 nM 11.5 ± 1.0  1 μM 10.7 ± 2.8  10μM 20.1 ± 3.1 100 μM 51.3 ± 2.6 S(+)DMS 100 nM 15.3 ± 7.7  1 μM 24.1 ±11.7  10 μM 47.0 ± 3.1 100 μM 76.9 ± 1.8

Relative potency can be expressed in terms of the concentration requiredto inhibit dopamine re-uptake by 50% (IC₅₀). The IC₅₀ values weredetermined graphically (see FIG. 18) and are shown below in Table 14.

TABLE 14 Concentrations Needed to Inhibit Dopamine Uptake by 50% AgentIC₅₀ Relative Potency Selegiline  ≈80 μM 1 R(−)DMS ≈100 μM 0.8 S(+)DMS ≈20 μM 4

The experiment described above was repeated in a concentration rangedesigned to more accurately describe IC₅₀ values and results are shownin FIG. 19. ID₅₀ values determined based upon the graph are shown inTable 15.

TABLE 15 Concentrations Needed to Inhibit Dopamine Uptake by 50% PotencyRelative to Compound ID₅₀ Selegiline S(+)DMS 11 μM 4.2 selegiline 46 μM1 R(−)DMS 54 μM 1.2

C. Conclusions

The results demonstrate that, at the appropriate concentration,selegiline and each of the enantiomers of DMS inhibit transport ofreleased dopamine at the neuronal synapse and enhance the relativeactivity of this neurotransmitter at the synapse. In this regard,S(+)DMS is more potent than selegiline which, in turn, is more potentthan R(−)DMS. Of the agents tested, S(+)DMS is the most preferred withregard to the treatment of hypodopaminergic conditions such as ADHD.

Example 9 Actions of the R(−) and S(+) enantiomers ofDesmethylselegiline (DMS) on Human Platelet MAO-B and Guinea Pig BrainMAO-B and MAO-A Activity

Human platelet MAO is comprised exclusively of the type-B isoform of theenzyme. In the present study, the in vitro and in vivo inhibition ofthis enzyme by the two enantiomers of DMS was determined and comparedwith inhibition due to selegiline. The present study also examined thetwo enantiomers of DMS for inhibitory activity with respect to the MAO-Aand MAO-B in guinea pig hippocampal tissue. Guinea pig brain tissue isan excellent animal model for studying brain dopamine metabolism, theenzyme kinetics of the multiple forms of MAO and the inhibitoryproperties of novel agents that interact with these enzymes. Themultiple forms of MAO in this animal species show similar kineticproperties to those found in human brain tissue. Finally, the testagents were administered to guinea pigs and the extent to which theymight act as inhibitors of brain MAO in vivo was assessed.

A. Method of Testing

In vitro: The test system utilized the in vitro conversion of specificsubstrates of MAO-A (¹⁴C-serotonin) and MAO-B (¹⁴C-phenylethylamine) byhuman platelets and/or guinea pig hippocampal homogenates. The rate ofconversion of each substrate was measured in the presence of S(+)DMS,R(−)DMS or selegiline and compared to the isozyme activity in theabsence of these agents. A percent inhibition was calculated from thesevalues. Potency was evaluated by comparing the concentration of eachagent which caused a 50% inhibition (IC₅₀ value).

In vivo: R(−)DMS, S(+)DMS or selegiline was administered in vivosubcutaneously (sc), once a day for 5 days prior to sacrifice,preparation of enzyme hippocampal homogenates, and the in vitro assay ofMAO-A and MAO-B activity. These experiments were performed todemonstrate that the DMS enantiomers were capable of entering braintissue and inhibiting MAO activity.

B. Results

MAO-B Inhibitory Activity In Vitro

Results for MAO-B inhibition are shown in Tables 16 and 17. IC₅₀ valuesfor MAO-B inhibition and potency as compared to selegiline is shown inTable 18.

TABLE 16 MAO-B Inhibition in Human Platelets % Inhibition AgentConcentration {overscore (x)} ± SEM Selegiline  0.3 nM  8.3 ± 3.4   5 nM50.3 ± 8.7   10 nM 69.0 ± 5.5   30 nM 91.0 ± 1.4  100 nM 96.0 ± 1.6  300nM 96.0 ± 1.6   1 μM 96.6 ± 1.6 R(−)DMS  100 nM 14.3 ± 3.6  300 nM 42.1± 4.0   1 μM 76.9 ± 1.47   3 μM 94.4 ± 1.4   10 μM 95.8 ± 1.4   3 μM95.7 ± 2.3 S(+)DMS  300 nM  6.4 ± 2.8   1 μM 11.1 ± 1.0   3 μM 26.6 ±1.9   10 μM 42.3 ± 2.3   30 μM 68.2 ± 2.34  100 μM 83.7 ± 0.77   1 mM94.2 ± 1.36

TABLE 17 MAO-B Inhibition in Guinea Pig Hippocampus % Inhibition AgentConcentration {overscore (x)} ± SEM Selegiline  0.3 nM 28.3 ± 8.7   5 nM81.2 ± 2.6   10 nM 95.6 ± 1.3   30 nM 98.5 ± 0.5  100 nM 98.8 ± 0.5  300nM 98.8 ± 0.5   1 μM 99.1 ± 0.45 R(−)DMS  100 nM 59.4 ± 9.6  300 nM 86.2± 4.7   1 μM 98.2 ± 0.7   3 μM 98.4 ± 0.95   10 μM 99.1 ± 0.45   30 μM99.3 ± 0.40 S(+)DMS  300 nM 18.7 ± 2.1   1 μM 44.4 ± 6.4   3 μM 77.1 ±6.0   10 μM 94.2 ± 1.9   30 μM 98.3 ± 0.6  100 μM 99.3 ± 0.2   1 mM 99.9± 0.1

TABLE 18 IC₅₀ Values for the Inhibition of MAO-B Guinea Pig TreatmentHuman Platelets Hippocampal Cortex Selegiline   5 nM (1)   1 nM (1)R(−)DMS  400 nM (80)  60 nM (60) S(+)DMS 1400 nM (2800) 1200 nM (1200) () = reduction in potency compared to selegiline

As observed, R(−)DMS was 20-35 times more potent than S(+)DMS as anMAO-B inhibitor and both enantiomers were less potent than selegiline.

MAO-A Inhibitory Activity In Vitro

Results obtained from experiments examining the inhibition of MAO-A inguinea pig hippocampus are summarized in Table 19. The IC₅₀ values forthe two enantiomers of DMS and for selegiline are shown in Table 20.

TABLE 19 MAO-A Inhibition in Guinea Pig Hippocampus % Reduction AgentConcentration {overscore (x)} ± SEM Selegiline 300 nM 11.95 ± 2.4  1 μM 22.1 ± 1.2  3 μM  53.5 ± 2.7  10 μM  91.2 ± 1.16 100 μM  98.1 ± 1.4  1mM  99.8 ± 0.2 R(−)DMS 300 nM  4.8 ± 2.1  1 μM  4.2 ± 1.5  3 μM  10.5 ±2.0  10 μM  19.0 ± 1.3 100 μM  64.2 ± 1.5  1 mM  96.5 ± 1.2 S(+)DMS  1μM  3.3 ± 1.5  3 μM  4.3 ± 1.0  10 μM  10.5 ± 1.47 100 μM  48.4 ± 1.8  1mM  92.7 ± 2.5  10 mM  99.6 ± 0.35

TABLE 20 IC₅₀ Values for the Inhibition of MAO-A IC₅₀ for MAO-A inGuinea Pig Treatment Hippocampal Cortex Selegiline  2.5 μM (1)  R(−)DMS 50.0 μM (20) S(+)DMS 100.0 μM (40) ( ) = reduction in potency comparedto selegiline

R(−)DMS was twice as potent as S(+)DMS as an MAO-A inhibitor and bothwere 20-40 times less potent than selegiline. Moreover, each of theseagents were 2-3 orders of magnitude, i.e., 100 to 1000 times, lesspotent as inhibitors of MAO-A than inhibitors of MAO-B in hippocampalbrain tissue. Therefore, selegiline and each enantiomer of DMS can beclassified as selective MAO-B inhibitors in brain tissue.

Results of In Vivo Experiments

Each enantiomer of DMS was administered in vivo by subcutaneousinjection once a day for five consecutive days, and inhibition of brainMAO-B activity was then determined. In preliminary studies, selegilinewas found to have an ID₅₀ of 0.03 mg/kg and both R(−)DMS and S(+)DMSwere determined to be about 10 times less potent. More recent studies,performed on a larger group of animals, indicates that R(−)DMS isactually about 25 times less potent than selegiline as an inhibitor ofMAO-B and that S(+)DMS is about 50 times less potent. Results are shownin FIG. 20 and ID₅₀ values are summarized in Table 21.

TABLE 21 ID₅₀ Values for Brain MAO-B Following 5 Days of AdministrationID₅₀ for MAO-B in Guinea Treatment Pig Hippocampal Cortex Selegiline0.008 mg/kg R(−)DMS  0.20 mg/kg S(+)DMS  0.50 mg/kg

This experiment demonstrates that the enantiomers of DMS penetrate theblood brain-barrier and inhibit brain MAO-B after in vivoadministration. It also demonstrates that the potency differences as anMAO-B inhibitor observed in vitro between each of the DMS enantiomersand selegiline are substantially reduced under in vivo conditions.

In experiments examining the effect of 5 s.c. treatments on MAO-Aactivity in guinea pig cortex (hippocampus), it was found thatselegiline administration at a dose of 1.0 mg/kg resulted in a 36.1%inhibition of activity. R(−)DMS resulted in an inhibition of 29.8% whenadministered at a dose of 3.0 mg/kg. S(+)DMS administration did notcause any observable inhibition at the highest dose tested (10 mg/kg)indicating that it has significantly less cross reactivity potential.

C. Conclusions

In vitro, R(−)DMS and S(+)DMS both exhibit activity as MAO-B and MAO-Ainhibitors. Each enantiomer was selective for MAO-B. S(+)DMS was lesspotent than R(−)DMS and both enantiomers of DMS were less potent thanselegiline in inhibiting both MAO-A and MAO-B.

In vivo, both enantiomers demonstrated activity in inhibiting MAO-B,indicating that these enantiomers are able to pass through theblood-brain barrier. The ability of these agents to inhibit MAO-Bsuggests that these agents may be of value as therapeutics forhypodopaminergic diseases such as ADHD and dementia.

Example 10 In Vivo Neuroprotection by the Enantiomers ofDesmethylselegiline

The ability of the enantiomers of DMS to prevent neurologicaldeterioration was examined by administering the agents to the wobblermouse, an animal model of motor neuron diseases, particularlyamyotrophic lateral sclerosis (ALS). Wobbler mice exhibit progressivelyworsening forelimb weakness, gait disturbances, and flexion contractionsof the forelimb muscles.

A. Test Method

A 0.1 mg/kg dose of R(−)DMS, S(+)DMS or placebo was administered towobbler mice by daily intra-peritoneal injection for a period of 30 daysin a randomized, double-blind study. At the end of this time mice wereexamined for grip strength, running time, resting locomotive activityand graded for semi-quantitative paw posture abnormalities, andsemi-quantitative walking abnormalities. The investigators who preparedand administered the test drugs to the animals were different than thosewho analyzed behavioral changes.

Assays and grading were performed essentially as described in Mitsumotoet al., Ann. Neurol. 36:142-148 (1994). Grip strength of the front pawsof a mouse was determined by allowing the animal to grasp a wire withboth paws. The wire was connected to a gram dynamometer and traction isapplied to the tail of the mouse until the animal is forced to releasethe wire. The reading on the dynamometer at the point of release istaken as a measure of grip strength.

Running time is defined as the shortest time necessary to traverse aspecified distance, e.g. 2.5 feet and the best time of several trials isrecorded.

Paw posture abnormalities are graded on a scale based upon the degree ofcontraction and walking abnormalities are graded on a scale ranging fromnormal walking to an inability to support the body using the paws.

Locomotive activity is determined by transferring animals to anexamination area in which the floor is covered with a square grid.Activity is measured by the number of squares traversed by a mouse in aset time interval, e.g., 9 minutes.

B. Results

At the beginning of the study, none of the groups were different in anyvariables, indicating that the three groups were comparative at thebaseline. Weight gain was identical in all three groups, suggesting thatno major side effects occurred in any animals. Table 22 summarizesdifferences that were observed in the mean grip strength of the testanimals:

TABLE 22 Mean Grip Strength in Wobbler Mice Treated with R(−) or S(+)DMSTreatment N Grip Strength (gm) Control (placebo) 10  9 (0-15) R(−)DMS 920 (0-63) S(+)DMS 9 14 (7-20) N = number of animals analyzed

Grip strength dropped markedly at the end of the first week in allanimals. At the end of the study, grip strength was the least in controlanimals. The variability in grip strength in the treated animal groupsprevented a meaningful statistical analysis of this data, however, at adose of 0.1 mg/kg, the mean grip strength measured in the DMS-treatedanimals was greater than for the controls. These results suggest thatthe dose may have been too low, and that a higher dose study should beperformed.

Running time, resting locomotive activity, semiquantitative paw postureabnormality grading, and semi-quantitative walking abnormality gradingwere also tested. None of these tests, however, showed any differenceamong the three groups tested.

Example 11 Immune System Restoration by R(−)DMS and S(+)DMS

There is an age-related decline in immunological function that occurs inanimals and humans which makes older individuals more susceptible toinfectious disease and cancer. U.S. Pat. Nos. 5,276,057 and 5,387,615suggest that selegiline is useful in the treatment of immune systemdysfunction. The present experiments were undertaken to determinewhether R(−)DMS and S(+) are also useful in the treatment of suchdysfunction. It should be recognized that an ability to bolster apatient's normal immunological defenses would be beneficial in thetreatment of a wide variety of acute and chronic diseases includingcancer, AIDS, and both bacterial and viral infections.

A. Test Procedure

The present experiments utilized a rat model to examine the ability ofR(−)DMS and S(+)DMS to restore immunological function. Rats were dividedinto the following experimental groups:

1) young rats (3 months old, no treatment);

2) old rats (18-20 months old, no treatment);

3) old rats injected with saline;

4) old rats treated with selegiline at a dosage of 0.25 mg/kg bodyweight;

5) old rats treated with selegiline at a dosage of 1.0 mg/kg bodyweight;

6) old rats treated with R(−)DMS at a dosage of 0.025 mg/kg body weight;

7) old rats treated with R(−)DMS at a dosage of 0.25 mg/kg body weight;

8) old rats treated with R(−)DMS at a dosage of 1.0 mg/kg body weight;

9) old rats treated with S(+)DMS at a dosage of 1.0 mg/kg body weight.

Rats were administered saline or test agent ip, daily for 60 days. Theywere then maintained for an additional “wash out” period of 10 daysduring which time no treatment was given. At the end of this time,animals were sacrificed and their spleens were removed. The spleen cellswere then assayed for a variety of factors which are indicative ofimmune system function. Specifically, standard tests were employed todetermine the following:

1) in vitro production of γ-interferon by concanavalin A-stimulatedspleen cells;

2) in vitro concanavalin A-induced production of interleukin-2;

3) percentage of IgM positive spleen cells (IgM is a marker of Blymphocytes);

4) percentage of CD5 positive spleen cells (CD5 is a marker of Tlymphocytes).

B. Results

The effect of administration of selegiline, R(−)DMS and S(+)DMS onconcanavalin A-induced interferon production by rat spleen cells isshown in Tables 23 and 24. Table 23 shows that there is a sharp declinein cellular interferon production that occurs with age. Administrationof selegiline, R(−)DMS and S(+)DMS all led to a restoration ofγ-interferon levels with the most dramatic increases occurring atdosages of 1.0 mg/kg body weight.

TABLE 23 Effect of Age on T Cell Function* IL-2 IFN-γ Groups U/ml std.error U/ml std. error young 59.4 18.27 12297 6447 old 19.6 7.52 338 135*T cell activities were assessed after stimulation of rat spleen cellswith concanavalin A. TH, cytokines, IL-2 and IFN-γ were measured. youngvs. old, p = 0.0004

TABLE 24 Mean and % control IL-2 and IFN g IL-2 U/ml IFN-γ U/ml Groupsmean % control mean % control control* 19.64 100 351 100 control 41.22210 339 96 R(−)DMS 55.17 281 573 163 R(−)DMS 64.54 329 516 147 R(−)DMS43.7 223 2728 777 S(+)DMS 57.12 291 918 261 Sel 0.25 109.6 558 795 226Sel. 1.0 73.78 376 1934 550 *Old rats (22 months old) with no treatment

Table 24 shows the extent to which R(−)DMS, S(+)DMS and selegiline arecapable of restoring γ-interferon production in the spleen cells of oldrats. Interferon-γ is a cytokine associated with T cells that inhibitviral replication and regulate a variety of immunological functions. Itinfluences the class of antibodies produced by B-cells, up-regulatesclass I and class II MHC complex antigens and increases the efficiencyof macrophage-mediated killing of intracellular parasites.

Histological immunofluorescence studies show a dramatic loss ofinnervation in rat spleens with age. When rats are treated with R(−)DMS,there is a significant increase in innervation in the spleens of animalsand this increase occurs in a dose-response manner. S(+) DMS did notshow any effect on histological examination, despite a modest increasein interferon-γ production. IL-2 production was not enhanced bytreatment with R(−)DMS or S(+)DMS, suggesting that the effects of theseagents may be limited to IFN-γ production.

C. Conclusions

The results obtained with respect to histological examination, theproduction of interferon, and the percentage of IgM positive spleencells support the conclusion that the DMS enantiomers are capable of atleast partially restoring the age-dependent loss of immune systemfunction. The results observed with respect to IFN-γ are particularlyimportant. In both humans and animals, IFN-γ production is associatedwith the ability to successfully recover from infection with viruses andother pathogens. In addition, it appears that R(−)DMS and S(+)DMS willhave a therapeutically beneficial effect for diseases and conditionsmediated by weakened host immunity. This would include AIDS, response tovaccines, infectious diseases and adverse immunological effects causedby cancer chemotherapy.

Example 12 Examples of Dosage Forms

A. Desmethylselegiline Patch.

Dry Weight Basis Component (mg/cm²) Durotak ® 87-2194 90 parts by weightadhesive acrylic polymer Desmethylselegiline 10 parts by weight

The two ingredients are thoroughly mixed, cast on a film backing sheet(e.g., Scotchpak® 9723 polyester) and dried. The backing sheet is cutinto patches a fluoropolymer release liner (e.g.,Scotchpak® 1022) isapplied, and the patch is hermetically sealed in a foil pouch. One patchis applied daily to supply 1-10 mg of desmethylselegiline per 24 hoursin the treatment of conditions in a human produced by neuronaldegeneration or neuronal trauma.

B. Ophthalmic Solution

Desmethylselegiline (0.1 g) as the hydrochloride, 1.9 g of boric acid,and .004 g of phenyl mercuric nitrate are dissolved in sterile water qs100 ml. The mixture is sterilized and sealed. It can be usedophthalmologically in the treatment of conditions produced by neuronaldegeneration or neuronal trauma, as for example glaucomatous opticneuropathy and macular degeneration.

C. Intravenous Solution.

a 1% solution is prepared by dissolving 1 g of desmethylselegiline asthe HCl in sufficient 0.9% isotonic saline solution to provide a finalvolume of 100 ml. The solution is buffered to pH 4 with citric acid,sealed, and sterilized to provide a 1% solution suitable for intravenousadministration in the treatment of conditions produced by neuronaldegeneration or neuronal trauma.

D. Oral Dosage Form

Tablets and capsules containing desmethylselegiline are prepared fromthe following ingredients (mg/unit dose):

desmethylselegiline   1-5 microcrystalline cellulose 86 lactose 41.6citric acid 0.5-2 sodium citrate 0.1-2 magnesium stearate  0.4 with anapproximately 1:1 ratio of citric acid and sodium citrate.

Having now fully described the invention, it will be understood by thoseof skill in the art that the invention may be performed within a wideand equivalent range of conditions, parameters and the like, withouteffecting the spirit or scope of the invention or any embodimentthereof.

What is claimed is:
 1. A method of treating Multiple Sclerosis in asubject suffering from Multiple Sclerosis which comprises administeringto the subject a pharmaceutical composition comprising substantiallyenantiomerically pure R(−)-desmethylselegiline or a pharmaceuticallyacceptable salt thereof, wherein the composition comprises apharmaceutically acceptable carrier, employing a single or multipledosage regimen effective to treat the Multiple Sclerosis.
 2. The methodof claim 1, wherein the subject is human.
 3. The method of claim 1,wherein the R(−)-desmethylselegiline is administered in a free baseform.
 4. The method of claim 1, wherein the pharmaceutically acceptablesalt is a hydrochloride salt.
 5. The method of claim 1, wherein thedosage regimen of R(−)-desmethylselegiline administered to the subjectper day is at least about 0.015 mg/kg to about 3.0 mg/kg of thesubject's body weight, calculated on the basis of the free secondaryamine.
 6. The method of claim 1, wherein the dosage regimen ofR(−)-desmethylselegiline administered to the subject per day is at leastabout 0.025 mg/kg to about 1.0 mg/kg of the subject's body weight,calculated on the basis of the free secondary amine.
 7. The method ofclaim 1, wherein the dosage regimen of R(−)-desmethylselegilineadministered to the subject per day is at least about 0.1 mg/kg to about0.5 mg/kg of the subject's body weight, calculated on the basis of thefree secondary amine.
 8. The method of claim 1, comprising administeringthe pharmaceutical composition by an oral route of administration. 9.The method of claim 1, comprising administering the pharmaceuticalcomposition by a non-oral route of administration.
 10. The method ofclaim 9, wherein the non-oral route of administration is parenteral,intravenous, subcutaneous, or intra-peritoneal.
 11. The method of claim9, wherein the non-oral route of administration is transdermal.
 12. Themethod of claim 9, wherein the non-oral route of administration issublingual or buccal.
 13. A method of treating Multiple Sclerosis in asubject suffering from Multiple Sclerosis which comprises administeringto the subject a pharmaceutical composition comprising substantiallyenantiomerically pure S(+)-desmethylselegiline or a pharmaceuticallyacceptable salt thereof, wherein the composition comprises apharmaceutically acceptable carrier, employing a single or multipledosage regimen effective to treat the Multiple Sclerosis.
 14. The methodof claim 13, wherein the subject is human.
 15. The method of herein theS(+)-desmethylselegiline is administered in a free base form.
 16. Themethod of claim 13, wherein the pharmaceutically acceptable salt is ahydrochloride salt.
 17. The method of claim 13, wherein the dosageregimen of S(+)-desmethylselegiline administered to the subject per dayis at least about 0.015 mg/kg to about 10 mg/kg of the subject's bodyweight, calculated on the basis of the free secondary amine.
 18. Themethod of claim 13, wherein the dosage regimen ofS(+)-desmethylselegiline administered to the subject per day is at leastabout 0.015 mg/kg to about 0.5 mg/kg of the subject's body weightcalculated on the basis of the free secondary amine.
 19. The method ofclaim 13, wherein the dosage regimen of S(+)-desmethylselegilineadministered to the subject per day is at least about 0.1 mg/kg to about1.0 mg/kg of the subject's body weight, calculated on the basis of thefree secondary amine.
 20. The method of claim 13, comprisingadministering the pharmaceutical composition by an oral route ofadministration.
 21. The method of claim 13, comprising administering thepharmaceutical composition by a non-oral route of administration. 22.The method of claim 21, wherein the non-oral route of administration isparenteral, intravenous, subcutaneous or intra-peritoneal.
 23. Themethod of claim 21, wherein the non-oral route of administration istransdermal.
 24. The method of claim 21, wherein the non-oral route ofadministration is sublingual or buccal.
 25. A method of treatingMultiple Sclerosis in a subject suffering from Multiple Sclerosis whichcomprises administering to the subject a pharmaceutical compositioncomprising a desmethylselegiline mixture, wherein thedesmethylselegiline mixture comprises R(−)-desmethylselegiline or apharmaceutically acceptable salt thereof, and S(+)-desmethylselegilineor a pharmaceutically acceptable salt thereof, employing a single ormultiple dosage regimen effective to treat the Multiple Sclerosis. 26.The method of claim 25, wherein the subject is human.
 27. The method ofclaim 25, wherein the R(−)-desmethylselegiline is administered in a freebase form.
 28. The method of claim 25, wherein theS(+)-desmethylselegiline is administered in a free base form.
 29. Themethod of claim 25, wherein the pharmaceutically acceptable salt is ahydrochloride salt.
 30. The method of claim 25, wherein the dosageregimen of R(−)-desmethylselegiline and S(+)-desmethylselegilineadministered to the subject per day is at least about 0.015 mg/kg toabout 1.0 mg/kg of the subject's body weight, calculated on the basis ofthe free secondary amine.
 31. The method of claim 25, wherein the dosageregimen of R(−)-desmethylselegiline and S(+)-desmethylselegilineadministered to the subject per day is at least about 0.1 mg/kg to about0.5 mg/kg of the subject's body weight, calculated on the basis of thefree secondary amine.
 32. The method of claim 25, comprisingadministering the pharmaceutical composition by an oral route ofadministration.
 33. The method of claim 25, comprising administering thepharmaceutical composition by a non-oral route of administration. 34.The method of claim 25, wherein the non-oral route of administration isparenteral, intravenous, subcutaneous or intra-peritoneal.
 35. Themethod of claim 25, wherein the non-oral route of administration istransdermal.
 36. The method of claim 25, wherein the non-oral route ofadministration is sublingual or buccal.